Her3 protein srm/mrm assay

ABSTRACT

The current disclosure provides for specific peptides, and derived ionization characteristics of the peptides, from the Receptor Tyrosine-Protein Kinase erbB-3, or Her3, that are particularly advantageous for quantifying the Her3 protein directly in biological samples that have been fixed in formalin by the method of Selected Reaction Monitoring (SRM) mass spectrometry, or what can also be termed as Multiple Reaction Monitoring (MRM) mass spectrometry. Such biological samples are chemically preserved and fixed wherein said biological sample is selected from tissues and cells treated with formaldehyde containing agents/fixatives including formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks, and tissue culture cells that have been formalin fixed and or paraffin embedded. A protein sample is prepared from said biological sample using the Liquid Tissue™ reagents and protocol and the Her3 protein is quantitated in the Liquid Tissue™ sample by the method of SRM/MRM mass spectrometry by quantitating in the protein sample at least one or more of the peptides described. These peptides can be quantitated if they reside in a modified or an unmodified form. An example of a modified form of a Her3 peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence.

This application claims the benefit of U.S. Provisional Application No.61/428,147, filed Dec. 29, 2010, entitled “Her3 Protein SRM/MRM Assay”the contents of which are hereby incorporated by reference in theirentirety.

INTRODUCTION

Specific peptides derived from subsequences of the ReceptorTyrosine-Protein Kinase erbB-3 protein and which will be referred to asHer3, and which can also be referred to as the proto-oncogene-likeprotein c-ErbB-3, the tyrosine kinase-type cell surface receptor Her3,and the ERBB3 protein are provided. The peptide sequence andfragmentation/transition ions for each peptide are particularly usefulin a mass spectrometry-based Selected Reaction Monitoring (SRM)assay(s), which can also be referred to as a Multiple ReactionMonitoring (MRM) assay(s), hereinafter referred to as SRM/MRM assay(s).The use of one such peptide for SRM/MRM quantitative analysis of theHer3 protein is described.

This SRM/MRM assay can be used to detect the presence and to measurerelative or absolute quantitative levels of one or more of the specificpeptides from the Her3 protein and therefore provide a means ofmeasuring the amount of the Her3 protein in a given protein preparationobtained from a biological sample by mass spectrometry.

The SRM/MRM assays described herein can measure Her3 peptides directlyin complex protein lysate samples prepared from cells procured frompatient tissue samples, such as formalin fixed cancer patient tissue.Methods of preparing protein samples from formalin-fixed tissue aredescribed in U.S. Pat. No. 7,473,532, the contents of which are herebyincorporated by references in their entirety. The methods described inthat patent may conveniently be carried out using Liquid Tissue™reagents and protocol available from Expression Pathology Inc.(Rockville, Md.).

Formaldehyde/formalin fixation of tissues surgically removed from cancerpatients is the accepted convention in pathology practice. As a result,formaldehyde/formalin fixed paraffin embedded tissue is the most widelyavailable form of tissues from those patients. Formaldehyde/formalinfixation typically employs aqueous solutions of formaldehyde referred toas formalin. “100%” formalin consists of a saturated solution offormaldehyde (about 40% formaldehyde by volume or 37% by mass) in water,with a small amount of stabilizer, usually methanol to limit oxidationand degree of polymerization. The most common way in which tissue ispreserved is to soak whole tissue for extended periods of time (8 hoursto 48 hours) in aqueous formaldehyde, commonly termed 10% neutralbuffered formalin, followed by embedding the fixed whole tissue inparaffin wax for long term storage at room temperature. Thus molecularanalytical methods to analyze formalin fixed cancer tissue will be themost accepted and heavily utilized methods for analysis of cancerpatient tissue.

Results from the SRM/MRM assay can be used to correlate accurate andprecise quantitative levels of the Her3 protein within the specifictissue samples (e.g., cancer tissue sample) of the patient or subjectfrom whom the tissue (biological sample) was collected and preserved.This not only provides diagnostic information about the cancer, but alsopermits a physician or other medical professional to determineappropriate therapy for the patient. Such an assay that providesdiagnostically and therapeutically important information about levels ofprotein expression in a diseased tissue or other patient sample istermed a companion diagnostic assay. For example, such an assay can bedesigned to diagnose the stage or degree of a cancer and determine atherapeutic agent to which a patient is most likely to respond.

SUMMARY

The assays described herein measure relative or absolute levels ofspecific unmodified peptides from the Her3 protein and also can measureabsolute or relative levels of specific modified peptides from the Her3protein. Examples of modifications include phosphorylated amino acidresidues and glycosylated amino acid residues that are present on thepeptides.

Relative quantitative levels of the Her3 protein are determined by theSRM/MRM methodology, for example by comparing SRM/MRM signature peakareas (e.g., signature peak area or integrated fragment ion intensity)of an individual Her3 peptide in different samples (e.g., a controlsample and an sample prepared from a patient's tissue). Alternatively,it is possible to compare multiple SRM/MRM signature peak areas formultiple Her3 signature peptides, where each peptide has its ownspecific SRM/MRM signature peak, to determine the relative Her3 proteincontent in one biological sample with the Her3 protein content in one ormore additional or different biological samples. In this way, the amountof a particular peptide, or peptides, from the Her3 protein, andtherefore the amount of the Her3 protein, is determined relative to thesame Her3 peptide, or peptides, across 2 or more biological samplesunder the same experimental conditions. In addition, relativequantitation can be determined for a given peptide, or peptides, fromthe Her3 protein within a single sample by comparing the signature peakarea for that peptide by SRM/MRM methodology to the signature peak areafor another and different peptide, or peptides, from a differentprotein, or proteins, within the same protein preparation from thebiological sample. In this way, the amount of a particular peptide fromthe Her3 protein, and therefore the amount of the Her3 protein, isdetermined relative one to another within the same sample. Theseapproaches generate quantitation of an individual peptide, or peptides,from the Her3 protein to the amount of another peptide, or peptides,between samples and within samples wherein the amounts as determined bypeak area are relative one to another, regardless of the absolute weightto volume or weight to weight amounts of the Her3 peptide in the proteinpreparation from the biological sample. Relative quantitative data aboutindividual signature peak areas between different samples are normalizedto the amount of protein analyzed per sample. Relative quantitation canbe performed across many peptides from multiple proteins and the Her3protein simultaneously in a single sample and/or across many samples togain insight into relative protein amounts, one peptide/protein withrespect to other peptides/proteins.

Absolute quantitative levels of the Her3 protein are determined by, forexample, the SRM/MRM methodology whereby the SRM/MRM signature peak areaof an individual peptide from the Her3 protein in one biological sampleis compared to the SRM/MRM signature peak area of a known amount of a“spiked” internal standard. In one embodiment, the internal standard isa synthetic version of the same exact Her3 peptide that contains one ormore amino acid residues labeled with one or more heavy isotopes. Suchisotope labeled internal standards are synthesized so mass spectrometryanalysis generates a predictable and consistent SRM/MRM signature peakthat is different and distinct from the native Her3 peptide signaturepeak and which can be used as a comparator peak. Thus when the internalstandard is spiked in known amounts into a protein or peptidepreparation from a biological sample in known amounts and analyzed bymass spectrometry, the SRM/MRM signature peak area of the native peptideis compared to the SRM/MRM signature peak area of the internal standardpeptide, and this numerical comparison indicates either the absolutemolarity and/or absolute weight of the native peptide present in theoriginal protein preparation from the biological sample. Absolutequantitative data for fragment peptides are displayed according to theamount of protein analyzed per sample. Absolute quantitation can beperformed across many peptides, and thus proteins, simultaneously in asingle sample and/or across many samples to gain insight into absoluteprotein amounts in individual biological samples and in entire cohortsof individual samples.

The SRM/MRM assay method can be used to aid diagnosis of the stage ofcancer, for example, directly in patient-derived tissue, such asformalin fixed tissue, and to aid in determining which therapeutic agentwould be most advantageous for use in treating that patient. Cancertissue that is removed from a patient either through surgery, such asfor therapeutic removal of partial or entire tumors, or through biopsyprocedures conducted to determine the presence or absence of suspecteddisease, is analyzed to determine whether or not a specific protein, orproteins, and which forms of proteins, are present in that patienttissue. Moreover, the expression level of a protein, or multipleproteins, can be determined and compared to a “normal” or referencelevel found in healthy tissue. Normal or reference levels of proteinsfound in healthy tissue may be derived from, for example, the relevanttissues of one or more individuals that do not have cancer.Alternatively, normal or reference levels may be obtained forindividuals with cancer by analysis of relevant tissues not affected bythe cancer.

Assays of protein levels (e.g., Her3 levels) can also be used todiagnose the stage of cancer in a patient or subject diagnosed withcancer by employing the Her3 levels. Levels or amounts of proteins orpeptides can be defined as the quantity expressed in moles, mass orweight of a protein or peptide determined by the SRM/MRM assay. Thelevel or amount may be normalized to the total level or amount ofprotein or another component in the lysate analyzed (e.g., expressed inmicromoles/microgram of protein or micrograms/microgram of protein). Inaddition, the level or amount of a protein or peptide may be determinedon volume basis, expressed, for example, in micromolar ornanograms/microliter. The level or amount of protein or peptide asdetermined by the SRM/MRM assay can also be normalized to the number ofcells analyzed. Information regarding Her3 can thus be used to aid indetermining stage or grade of a cancer by correlating the level of theHer3 protein (or fragment peptides of the Her3 protein) with levelsobserved in normal tissues.

Once the stage and/or grade, and/or Her3 protein expressioncharacteristics of the cancer has been determined, that information canbe matched to a list of therapeutic agents (chemical and biological)developed to specifically treat cancer tissue that is characterized by,for example, abnormal expression of the protein or protein(s) (e.g.,Her3) that were assayed. Matching information from a Her3 protein assayto a list of therapeutic agents that specifically targets, for example,the Her3 protein or cells/tissue expressing the protein, defines whathas been termed a personalized medicine approach to treating disease.The assay methods described herein form the foundation of a personalizedmedicine approach by using analysis of proteins from the patient's owntissue as a source for diagnostic and treatment decisions.

Certain embodiments of the invention are described below.

-   -   1. A method for measuring the level of Receptor Tyrosine-Protein        Kinase erbB-3 (Her3) protein in a biological sample, comprising        detecting and/or quantifying the amount of one or more modified        or unmodified Her3 fragment peptides in a protein digest        prepared from said biological sample using mass spectrometry;        and calculating the level of modified or unmodified Her3 protein        in said sample; and wherein said level is a relative level or an        absolute level.    -   2. The method of embodiment 1, further comprising the step of        fractionating said protein digest prior to detecting and/or        quantifying the amount of one or more modified or unmodified        Her3 fragment peptides.    -   3. The method of embodiment 2, wherein said fractionating step        is selected from the group consisting of gel electrophoresis,        liquid chromatography, capillary electrophoresis, nano-reversed        phase liquid chromatography, high performance liquid        chromatography, or reverse phase high performance liquid        chromatography.    -   4. The method of any of embodiments 1-3, wherein said protein        digest of said biological sample is prepared by the Liquid        Tissue™ protocol.    -   5. The method of any of embodiments 1-3, wherein said protein        digest comprises a protease digest.    -   6. The method of embodiment 5, wherein said protein digest        comprises a trypsin digest.    -   7. The method of any of embodiments 1-6, wherein said mass        spectrometry comprises tandem mass spectrometry, ion trap mass        spectrometry, triple quadrupole mass spectrometry, MALDI-TOF        mass spectrometry, MALDI mass spectrometry, and/or time of        flight mass spectrometry.    -   8. The method of embodiment 7, wherein the mode of mass        spectrometry used is Selected Reaction Monitoring (SRM),        Multiple Reaction Monitoring (MRM), and/or multiple Selected        Reaction Monitoring (mSRM).    -   9. The method of any of embodiments 1 to 8, wherein the Her3        fragment peptide comprises an amino acid sequence as set forth        as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID        NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and        SEQ ID NO:10.    -   10. The method of any of embodiments 1-9, wherein the biological        sample is a blood sample, a urine sample, a serum sample, an        ascites sample, a sputum sample, lymphatic fluid, a saliva        sample, a cell, or a solid tissue.    -   11. The method of embodiment 10, wherein the tissue is formalin        fixed tissue.    -   12. The method of embodiment 10 or 11, wherein the tissue is        paraffin embedded tissue.    -   13. The method of embodiment 10, wherein the tissue is obtained        from a tumor.    -   14. The method of embodiment 13, wherein the tumor is a primary        tumor.    -   15. The method of embodiment 13, wherein the tumor is a        secondary tumor.    -   16. The method of any of embodiments 1-15, further comprising        quantifying a modified or unmodified Her3 fragment peptide.    -   17. The method of embodiment 16, wherein quantifying the Her3        fragment peptide comprises comparing an amount of one or more        Her3 fragment peptides comprising an amino acid sequence of        about 8 to about 45 amino acid residues of Her3 as shown in SEQ        ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ        ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10        in one biological sample to the amount of the same Her3 fragment        peptide in a different and separate biological sample.    -   18. The method of embodiment 17, wherein quantifying one or more        Her3 fragment peptides comprises determining the amount of the        each of the Her3 fragment peptides in a biological sample by        comparison to an added internal standard peptide of known        amount, wherein each of the Her3 fragment peptides in the        biological sample is compared to an internal standard peptide        having the same amino acid sequence.    -   19. The method of embodiment 18, wherein the internal standard        peptide is an isotopically labeled peptide.    -   20. The method of embodiment 19, wherein the isotopically        labeled internal standard peptide comprises one or more heavy        stable isotopes selected from 18O, 17O, 34S, 15N, 13C, 2H or        combinations thereof.    -   21. The method of any of embodiments 1-20, wherein detecting        and/or quantifying the amount of one or more modified or        unmodified Her3 fragment peptides in the protein digest        indicates the presence of modified or unmodified Her3 protein        and an association with cancer in the subject.    -   22. The method of embodiment 21, further comprising correlating        the results of said detecting and/or quantifying the amount of        one or more modified or unmodified Her3 fragment peptides, or        the level of said Her3 protein to the diagnostic        stage/grade/status of the cancer.    -   23. The method of embodiment 22, wherein correlating the results        of said detecting and/or quantifying the amount of one or more        modified or unmodified Her3 fragment peptides, or the level of        said Her3 protein to the diagnostic stage/grade/status of the        cancer is combined with detecting and/or quantifying the amount        of other proteins or peptides from other proteins in a multiplex        format to provide additional information about the diagnostic        stage/grade/status of the cancer.    -   24. The method of any one of embodiments 1-23, further        comprising selecting for the subject from which said biological        sample was obtained a treatment based on the presence, absence,        or amount of one or more Her3 fragment peptides or the level of        Her3 protein.    -   25. The method of any one of embodiments 1-24, further        comprising administering to the patient from which said        biological sample was obtained a therapeutically effective        amount of a therapeutic agent, wherein the therapeutic agent        and/or amount of the therapeutic agent administered is based        upon amount of one or more modified or unmodified Her3 fragment        peptides or the level of Her3 protein.    -   26. The method of embodiments 24 and 25, wherein therapeutic        agents bind the Her3 protein and/or inhibit its biological        activity.    -   27. The method of embodiments 1-26, wherein the biological        sample is formalin fixed tumor tissue that has been processed        for quantifying the amount of one or more modified or unmodified        Her3 fragment peptides employing the Liquid Tissue™ protocol and        reagents.    -   28. The method of any of embodiments 1-27, wherein said one or        more modified or unmodified Her3 fragment peptides is two or        more, three or more, four or more, five or more, six or more,        eight or more, or nine or more of the peptides in Table 1.    -   29. The method of any of embodiments 1-28, comprising        quantifying the amount of the peptide in Table 2.    -   30. A composition comprising one or more, two or more, three or        more, four or more, five or more, six or more, eight or more, or        nine or more of the peptides in Table 1 or antibodies thereto.    -   31. The composition of embodiment 30 comprising the peptide of        Table 2 or an antibody thereto.    -   32. The composition of embodiments 30 or 31, wherein said        composition is substantially pure or free of other cellular        components selected from any combination of other proteins,        membranes lipids and/or nucleic acids.    -   33. The composition of any of embodiment 30-32, wherein said        peptides are isotopically labeled internal standard peptides        that comprises one or more, two or more, or three or more, heavy        stable isotopes selected from 18O, 17O, 34S, 15N, 13C, 2H or        combinations thereof.    -   34. The method of embodiments 1-29, further comprising assessing        and/or determining the level (amount) or sequence of one, two,        three, four or more nucleic acids in said protein digest.    -   35. The method of claim 34, wherein said nucleic acids have a        length greater than about 15, 20, 25, 30, 35, 40, 50, 60, 75, or        100 nucleotides in length.    -   36. The method of claim 35, wherein said nucleic acids have a        length less than about 150, 200, 250, 300, 350, 400, 500, 600,        750, 1,000, 2,000, 4,000, 5,000, 7,500, 10,000, 15,000, or        20,000 nucleotides in length.    -   37. The method of any of embodiments 34-36, wherein assessing        and/or determining the level (amount) or sequence comprises,        determining either the sequence of nucleotides in the nucleic        acids and/or a characteristic of the nucleic acids by any one or        more of: nucleic acid sequencing, conducting restriction        fragment polymorphism analysis, nucleic acid hybridization        identification of one or more deletions and/or insertions,        and/or determining the presence of mutations, including but not        limited to, single base pair polymorphisms, transitions and/or        transversions.

DETAILED DESCRIPTION

In principle, any predicted peptide derived from Her3 protein, preparedfor example by digesting with a protease of known specificity (e.g.trypsin), can be used as a surrogate reporter to determine the abundanceof Her3 protein in a sample using a mass spectrometry-based SRM/MRMassay. Similarly, any predicted peptide sequence containing an aminoacid residue at a site that is known to be potentially modified in Her3protein also might potentially be used to assay the extent ofmodification of Her3 protein in a sample.

Her3 fragment peptides may be generated by a variety of means includingby the use of the Liquid Tissue™ protocol provided in U.S. Pat. No.7,473,532. The Liquid Tissue™ protocol and reagents are capable ofproducing peptide samples suitable for mass spectroscopic analysis fromformalin fixed paraffin embedded tissue by proteolytic digestion of theproteins in the tissue/biological sample. In the Liquid Tissue™ protocolthe tissue/biological is maintained at elevated temperatures in a bufferfor an extended period of time (e.g., from about 80° C. to about 100° C.for a period of time from about 10 minutes to about 4 hours) to reverseor release protein cross-linking. The buffer employed is a neutralbuffer, (e.g., a Tris-based buffer, or a buffer containing a detergent)and advantageously is a buffer that does not interfere with massspectrometric analysis. Next the tissue/biological sample is treatedwith one or more proteases, including but not limited to trypsin,chymotrypsin, pepsin, and endoproteinase Lys-C for a time sufficient todisrupt the tissue and cellular structure of said biological sample andto liquefy said sample (e.g., a period of time from 30 minutes to 24hours at a temperature from 37° C. to 65° C.). The result of the heatingand proteolysis is a liquid, soluble, dilutable biomolecule lysate.

Once lysates are prepared peptides in the samples may subject to avariety of techniques that facilitate their analysis and measurement bymass spectrometry. In one embodiment, the peptides may be separated byan affinity technique, such as for example immunologically-basedpurification (e g, immunoaffinity chromatography), chromatography on ionselective media, or if the peptides are modified, by separation usingappropriate media, such as lectins for separation of carbohydratemodified peptides. In one embodiment, the SISCAPA method, which employsimmunological separation of peptides prior to mass spectrometricanalysis is employed. The SISCAPA technique is described, for example,in U.S. Pat. No. 7,632,686. In other embodiments, lectin affinitymethods (e.g., affinity purification and/or chromatography may be usedto separate peptides from a lysate prior to analysis by massspectrometry. Methods for separation of groups of peptides, includinglectin-based methods, are described, for example, in Geng et al., J.Chromatography B, 752:293-306 (2001). Immunoaffinity chromatographytechniques, lectin affinity techniques and other forms of affinityseparation and/or chromatography (e.g., reverse phase, size basedseparation, ion exchange) may be used in any suitable combination tofacilitate the analysis of peptides by mass spectrometry.

Surprisingly, it was found that many potential peptide sequences fromthe Her3 protein are unsuitable or ineffective for use in massspectrometry-based SRM/MRM assays for reasons that are not immediatelyevident. In particular it was found that many tryptic peptides from theHer3 protein could not be detected efficiently or at all in a LiquidTissue lysate from formalin fixed, paraffin embedded tissue. As it wasnot possible to predict the most suitable peptides for MRM/SRM assay, itwas necessary to experimentally identify modified and unmodifiedpeptides in actual Liquid Tissue™ lysates to develop a reliable andaccurate SRM/MRM assay for the Her3 protein. While not wishing to bebound by any theory, it is believed that some peptides might, forexample, be difficult to detect by mass spectrometry as they do notionize well or produce fragments distinct from other proteins, peptidesmay also fail to resolve well in separation (e.g., liquidchromatography), or adhere to glass or plastic ware. Accordingly, thosepeptides from the Her3 protein that can be detected in a Liquid Tissuelysate (e.g., the peptides in Tables 1 and 2) prepared from a formalinfixed tissue sample are the peptides for which SRM/MRM assays can beemployed in a Her3 SRM/MRM assay.

Her3 peptides found in various embodiments of this disclosure (e.g.,Tables 1 and 2) were derived from the Her3 protein by protease digestionof all the proteins within a complex Liquid Tissue™ lysate prepared fromcells procured from formalin fixed cancer tissue. Unless notedotherwise, in each instance the protease was trypsin. The Liquid Tissue™lysate was then analyzed by mass spectrometry to determine thosepeptides derived from the Her3 protein that are detected and analyzed bymass spectrometry. Identification of a specific preferred subset ofpeptides for mass-spectrometric analysis is based on: 1) experimentaldetermination of which peptide or peptides from a protein ionize in massspectrometry analyses of Liquid Tissue™ lysates; and 2) the ability ofthe peptide to survive the protocol and experimental conditions used inpreparing a Liquid Tissue™ lysate. This latter property extends not onlyto the amino acid sequence of the peptide but also to the ability of amodified amino acid residue within a peptide to survive in modified formduring the sample preparation.

TABLE 1 Peptide Peptide Sequence SEQ ID NO: 1 ELANEFTR SEQ ID NO: 2LAEVPDLLEK SEQ ID NO: 3 IYISANR SEQ ID NO: 4AFQGPGHQAPHVHY[Phosphoryl]AR SEQ ID NO: 5 SLEATDSAFDNPDY[Phosphoryl]WHSRSEQ ID NO: 6 DGGGPGGDY[Phosphoryl]AAMGACPAS EQGY[Phosphoryl]EEMRSEQ ID NO: 7 DGGGPGGDY[Phosphoryl]AAMGACPAS EQGYEEMR SEQ ID NO: 8DGGGPGGDYAAMGACPASEQGY [Phosphoryl]EEMR SEQ ID NO: 9DGGGPGGDYAAMGACPASEQGYEEMR SEQ ID NO: 10 ANDALQVLGLLFSLAR

TABLE 2 Pre- Mono cursor Tran- Peptide Isotopic Charge Precursor sitionIon SEQ ID sequence Mass State m/z m/z Type SEQ ID ANDALQVL 1700.96938 2850.9879761 876.5296 y8 NO: 10 GLLFSLAR 2 989.6137 y9 2 1088.682 y10 21216.741 y11 2 1329.825 y12 2 1400.862 y13

Protein lysates from cells procured directly from formalin(formaldehyde) fixed tissue were prepared using the Liquid Tissue™reagents and protocol that entails collecting cells into a sample tubevia tissue microdissection followed by heating the cells in the LiquidTissue™ buffer for an extended period of time. Once the formalin-inducedcross linking has been negatively affected, the tissue/cells are thendigested to completion in a predictable manner using a protease, as forexample including but not limited to the protease trypsin. Each proteinlysate is turned into a collection of peptides by digestion of intactpolypeptides with the protease. Each Liquid Tissue™ lysate was analyzed(e.g., by ion trap mass spectrometry) to perform multiple globalproteomic surveys of the peptides where the data was presented asidentification of as many peptides as could be identified by massspectrometry from all cellular proteins present in each protein lysate.An ion trap mass spectrometer or another form of a mass spectrometerthat is capable of performing global profiling for identification of asmany peptides as possible from a single complex protein/peptide lysateis employed. Ion trap mass spectrometers however may be the best type ofmass spectrometer for conducting global profiling of peptides. AlthoughSRM/MRM assay can be developed and performed on any type of massspectrometer, including a MALDI, ion trap, or triple quadrupole, themost advantageous instrument platform for SRM/MRM assay is oftenconsidered to be a triple quadrupole instrument platform.

Once as many peptides as possible were identified in a single MSanalysis of a single lysate under the conditions employed, then thatlist of peptides was collated and used to determine the proteins thatwere detected in that lysate. That process was repeated for multipleLiquid Tissue™ lysates, and the very large list of peptides was collatedinto a single dataset. That type of dataset can be considered torepresent the peptides that can be detected in the type of biologicalsample that was analyzed (after protease digestion), and specifically ina Liquid Tissue™ lysate of the biological sample, and thus includes thepeptides for specific proteins, such as for example the Her3 protein.

In one embodiment, the Her3 tryptic peptides identified as useful in thedetermination of absolute or relative amounts of the Her3 receptorinclude one or more, two or more, three or more, four or more, five ormore, six or more, eight or more, or nine or more of the peptides of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, each ofwhich are listed in Table 1. Each of those peptides was detected by massspectrometry in Liquid Tissue™ lysates prepared from formalin fixed,paraffin embedded tissue. Thus, each of the peptides in Table 1, or anycombination of those peptides (e.g., one or more, two or more, three ormore, four or more, five or more, six or more, eight or more, or nine ormore of those peptides recited in Table 1, and particularly combinationswith the peptide found in Table 2) are candidates for use inquantitative SRM/MRM assay for the Her3 protein in human biologicalsamples, including directly in formalin fixed patient tissue.

The Her3 tryptic peptides listed in Table 1 include those detected frommultiple Liquid Tissue™ lysates of multiple different formalin fixedtissues of different human organs including prostate, colon, and breast.Each of those peptides is considered useful for quantitative SRM/MRMassay of the Her3 protein in formalin fixed tissue. Further dataanalysis of these experiments indicated no preference is observed forany specific peptides from any specific organ site. Thus, each of thesepeptides is believed to be suitable for conducting SRM/MRM assays of theHer3 protein on a Liquid Tissue™ lysate from any formalin fixed tissueoriginating from any biological sample or from any organ site in thebody.

In one embodiment the peptides in Table 1, or any combination of thosepeptides (e.g., one or more, two or more, three or more, four or more,five or more, six or more, eight or more, or nine or more of thosepeptides recited in Table 1, and particularly combinations with thepeptide also found in Table 2) are assayed by methods that do not relyupon mass spectroscopy, including, but not limited to, immunologicalmethods (e.g., Western blotting or ELISA). Regardless of how informationdirected to the amount of the peptide(s) (absolute or relative) isobtained, the information may be employed in any of the methodsdescribed herein, including indicating (diagnosing) the presence ofcancer in a subject, determining the stage/grade/status of the cancer,providing a prognosis, or determining the therapeutics or treatmentregimen for a subject/patient.

Embodiments of the present disclosure include compositions comprisingone or more, two or more, three or more, four or more, five or more, sixor more, eight or more, or nine or more of the peptides in Table 1. Insome embodiments, the compositions comprise the peptide in Table 2.Compositions comprising peptides may include one or more, two or more,three or more, four or more, five or more, six or more, eight or more,or nine or more peptides that are isotopically labeled. Each of thepeptides may be labeled with one or more isotopes selected independentlyfrom the group consisting of: ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C, ²H orcombinations thereof. Compositions comprising peptides from the Her3protein, whether isotope labeled or not, do not need to contain all ofthe peptides from that protein (e.g., a complete set of trypticpeptides). In some embodiments the compositions do not contain one ormore, two or more, three or more, four or more, five or more, six ormore, eight or more, or nine or more peptides from Her3, andparticularly peptides appearing in Table 1 or Table 2. Compositionscomprising peptides may be in the form of dried or lyophized materials,liquid (e.g., aqueous) solutions or suspensions, arrays, or blots.

An important consideration when conducting an SRM/MRM assay is the typeof instrument that may be employed in the analysis of the peptides.Although SRM/MRM assays can be developed and performed on any type ofmass spectrometer, including a MALDI, ion trap, or triple quadrupole,presently the most advantageous instrument platform for SRM/MRM assay isoften considered to be a triple quadrupole instrument platform. Thattype of a mass spectrometer may be considered to be the most suitableinstrument for analyzing a single isolated target peptide within a verycomplex protein lysate that may consist of hundreds of thousands tomillions of individual peptides from all the proteins contained within acell.

In order to most efficiently implement SRM/MRM assay for each peptidederived from the Her3 protein it is desirable to utilize information inaddition to the peptide sequence in the analysis. That additionalinformation may be used in directing and instructing the massspectrometer (e.g. a triple quadrupole mass spectrometer), to performthe correct and focused analysis of specific targeted peptide(s), suchthat the assay may be effectively performed.

The additional information about target peptides in general, and aboutspecific Her3 peptides, may include one or more of the mono isotopicmass of each peptide, its precursor charge state, the precursor m/zvalue, the m/z transition ions, and the ion type of each transition ion.Additional peptide information that may be used to develop an SRM/MRMassay for the Her3 protein is shown by example for one (1) of the Her3peptides from the list in Table 1 and is shown in Table 2. Similaradditional information described for this one (1) Her3 peptide shown byexample in Table 2 may be prepared, obtained, and applied to theanalysis of the other peptides contained in Table 1.

The method described below was used to: 1) identify candidate peptidesfrom the Her3 protein that can be used for a mass spectrometry-basedSRM/MRM assay for the Her3 protein, 2) develop individual SRM/MRM assay,or assays, for target peptides from the Her3 protein in order tocorrelate and 3) apply quantitative assays to cancer diagnosis and/orchoice of optimal therapy.

Assay Method 1. Identification of SRM/MRM Candidate Fragment Peptidesfor the Her3 Protein

-   -   a. Prepare a Liquid Tissue™ protein lysate from a formalin fixed        biological sample using a protease or proteases, (that may or        may not include trypsin), to digest proteins    -   b. Analyze all protein fragments in the Liquid Tissue™ lysate on        an ion trap tandem mass spectrometer and identify all fragment        peptides from the Her3 protein, where individual fragment        peptides do not contain any peptide modifications such as        phosphorylations or glycosylations    -   c. Analyze all protein fragments in the Liquid Tissue™ lysate on        an ion trap tandem mass spectrometer and identify all fragment        peptides from the Her3 protein that carry peptide modifications        such as for example phosphorylated or glycosylated residues    -   d. All peptides generated by a specific digestion method from        the entire, full length Her3 protein potentially can be        measured, but preferred peptides used for development of the        SRM/MRM assay are those that are identified by mass spectrometry        directly in a complex Liquid Tissue™ protein lysate prepared        from a formalin fixed biological sample    -   e. Peptides that are specifically modified (phosphorylated,        glycosylated, etc.) in patient tissue and which ionize, and thus        can be detected, in a mass spectrometer when analyzing a Liquid        Tissue™ lysate from a formalin fixed biological sample are        identified as candidate peptides for assaying peptide        modifications of the Her3 protein        2. Mass Spectrometry Assay for Fragment Peptides from Her3        Protein    -   a. SRM/MRM assay on a triple quadrupole mass spectrometer for        individual fragment peptides identified in a Liquid Tissue™        lysate is applied to peptides from the Her3 protein        -   i. Determine optimal retention time for a fragment peptide            for optimal chromatography conditions including but not            limited to gel electrophoresis, liquid chromatography,            capillary electrophoresis, nano-reversed phase liquid            chromatography, high performance liquid chromatography, or            reverse phase high performance liquid chromatography        -   ii. Determine the mono isotopic mass of the peptide, the            precursor charge state for each peptide, the precursor m/z            value for each peptide, the m/z transition ions for each            peptide, and the ion type of each transition ion for each            fragment peptide in order to develop an SRM/MRM assay for            each peptide.        -   iii. SRM/MRM assay can then be conducted using the            information from (i) and (ii) on a triple quadrupole mass            spectrometer where each peptide has a characteristic and            unique SRM/MRM signature peak that precisely defines the            unique SRM/MRM assay as performed on a triple quadrupole            mass spectrometer    -   b. Perform SRM/MRM analysis so that the amount of the fragment        peptide of the Her3 protein that is detected, as a function of        the unique SRM/MRM signature peak area from an SRM/MRM mass        spectrometry analysis, can indicate both the relative and        absolute amount of the protein in a particular protein lysate.        -   i. Relative quantitation may be achieved by:            -   1. Determining increased or decreased presence of the                Her3 protein by comparing the SRM/MRM signature peak                area from a given Her3 peptide detected in a Liquid                Tissue™ lysate from one formalin fixed biological sample                to the same SRM/MRM signature peak area of the same Her3                fragment peptide in at least a second, third, fourth or                more Liquid Tissue™ lysates from least a second, third,                fourth or more formalin fixed biological samples            -   2. Determining increased or decreased presence of the                Her3 protein by comparing the SRM/MRM signature peak                area from a given Her3 peptide detected in a Liquid                Tissue™ lysate from one formalin fixed biological sample                to SRM/MRM signature peak areas developed from fragment                peptides from other proteins, in other samples derived                from different and separate biological sources, where                the SRM/MRM signature peak area comparison between the 2                samples for a peptide fragment are normalized to amount                of protein analyzed in each sample.            -   3. Determining increased or decreased presence of the                Her3 protein by comparing the SRM/MRM signature peak                area for a given Her3 peptide to the SRM/MRM signature                peak areas from other fragment peptides derived from                different proteins within the same Liquid Tissue™ lysate                from the formalin fixed biological sample in order to                normalize changing levels of Her3 protein to levels of                other proteins that do not change their levels of                expression under various cellular conditions.            -   4. These assays can be applied to both unmodified                fragment peptides and for modified fragment peptides of                the Her3 protein, where the modifications include but                are not limited to phosphorylation and/or glycosylation,                and where the relative levels of modified peptides are                determined in the same manner as determining relative                amounts of unmodified peptides.        -   ii. Absolute quantitation of a given peptide may be achieved            by comparing the SRM/MRM signature peak area for a given            fragment peptide from the Her3 protein in an individual            biological sample to the SRM/MRM signature peak area of an            internal fragment peptide standard spiked into the protein            lysate from the biological sample            -   1. The internal standard is a labeled synthetic version                of the fragment peptide from the Her3 protein that is                being interrogated. This standard is spiked into a                sample in known amounts, and the SRM/MRM signature peak                area can be determined for both the internal fragment                peptide standard and the native fragment peptide in the                biological sample separately, followed by comparison of                both peak areas            -   2. This can be applied to unmodified fragment peptides                and modified fragment peptides, where the modifications                include but are not limited to phosphorylation and/or                glycosylation, and where the absolute levels of modified                peptides can be determined in the same manner as                determining absolute levels of unmodified peptides.

3. Apply Fragment Peptide Quantitation to Cancer Diagnosis and Treatment

-   -   a. Perform relative and/or absolute quantitation of fragment        peptide levels of the Her3 protein and demonstrate that the        previously-determined association, as well understood in the        field of cancer, of Her3 protein expression to the        stage/grade/status of cancer in patient tumor tissue is        confirmed    -   b. Perform relative and/or absolute quantitation of fragment        peptide levels of the Her3 protein and demonstrate correlation        with clinical outcomes from different treatment strategies,        wherein this correlation has already been demonstrated in the        field or can be demonstrated in the future through correlation        studies across cohorts of patients and tissue from those        patients. Once either previously established correlations or        correlations derived in the future are confirmed by this assay        then the assay method can be used to determine optimal treatment        strategy.

Assessment of Her3 protein levels in tissues based on analysis offormalin fixed patient-derived tissue can provide diagnostic,prognostic, and therapeutically-relevant information about eachparticular patient. In one embodiment, this disclosure describes amethod for measuring the level of the Her3 protein in a biologicalsample, comprising detecting and/or quantifying the amount of one ormore modified or unmodified Her3 fragment peptides in a protein digestprepared from said biological sample using mass spectrometry; andcalculating the level of modified or unmodified Her3 protein in saidsample; and wherein said level is a relative level or an absolute level.In a related embodiment, quantifying one or more Her3 fragment peptidescomprises determining the amount of the each of the Her3 fragmentpeptides in a biological sample by comparison to an added internalstandard peptide of known amount, wherein each of the Her3 fragmentpeptides in the biological sample is compared to an internal standardpeptide having the same amino acid sequence. In some embodiments theinternal standard is an isotopically labeled internal standard peptidecomprises one or more heavy stable isotopes selected from ¹⁸O, ¹⁷O, ³⁴S,¹⁵N, ¹³C, ²H or combinations thereof.

The method for measuring the level of the Her3 protein in a biologicalsample described herein (or fragment peptides as surrogates thereof) maybe used as a diagnostic indicator of cancer in a patient or subject. Inone embodiment, the results from measurements of the level of the Her3protein may be employed to determine the diagnostic stage/grade/statusof a cancer by correlating (e.g., comparing) the level of Her3 receptorfound in a tissue with the level of that protein found in normal and/orcancerous or precancerous tissues.

Because both nucleic acids and protein can be analyzed from the sameLiquid Tissue biomolecular preparation it is possible to generateadditional information about disease diagnosis and drug treatmentdecisions from the same sample. For example, the Her3 protein is atyrosine kinase receptor that is capable of stimulating uncontrolledcell growth (cancer) by activation of specific cell signal proteinpathways. If Her3 is expressed by certain cells to at increased levels,when assayed by SRM the data can provide information about the state ofthe cells and their potential for uncontrolled growth, potential drugresistance and the development of cancers can be obtained. At the sametime, information about the status of the Her3 gene and/or the nucleicacids and proteins it encodes (e.g., mRNA molecules and their expressionlevels or splice variations) can be obtained from nucleic acids presentin the same biomolecular preparation. For example information about Her3and/or one, two, three, four or more additional proteins may be assessedby examining the nucleic acids encoding those proteins. Those nucleicacids can be examined, for example, by one or more sequencing methods,conducting restriction fragment polymorphism analysis, identification ofdeletions, insertions, and/or determining the presence of mutations,including but not limited to, single base pair polymorphisms,transitions and/or transversions.

The above description and exemplary embodiments of methods andcompositions are illustrative of the scope of the present disclosure.Because of variations which will be apparent to those skilled in theart, however, the present disclosure is not intended to be limited tothe particular embodiments described above

1. A method for measuring the level of Receptor Tyrosine-Protein KinaseerbB-3 (Her3) protein in a biological sample, comprising detecting andquantifying the amount of one or more modified or unmodified Her3fragment peptides in a protein digest prepared from said biologicalsample using mass spectrometry; and calculating the level of modified orunmodified Her3 protein in said sample; and wherein said level is arelative level or an absolute level.
 2. The method of claim 1, furthercomprising the step of fractionating said protein digest prior todetecting and/or quantifying the amount of one or more modified orunmodified Her3 fragment peptides.
 3. The method of claim 2, whereinsaid fractionating step is selected from the group consisting of gelelectrophoresis, liquid chromatography, capillary electrophoresis,nano-reversed phase liquid chromatography, high performance liquidchromatography, or reverse phase high performance liquid chromatography.4. The method of any of claim 1 wherein said protein digest of saidbiological sample is prepared by the Liquid Tissue™ protocol.
 5. Themethod of any of claim 1, wherein said protein digest comprises aprotease digest.
 6. The method of claim 5, wherein said protein digestcomprises a trypsin digest.
 7. The method of claim 1, wherein said massspectrometry comprises tandem mass spectrometry, ion trap massspectrometry, triple quadrupole mass spectrometry, MALDI-TOF massspectrometry, MALDI mass spectrometry, and/or time of flight massspectrometry.
 8. The method of claim 7, wherein the mode of massspectrometry used is Selected Reaction Monitoring (SRM), MultipleReaction Monitoring (MRM), and/or multiple Selected Reaction Monitoring(mSRM).
 9. The method of claim 1, wherein the Her3 fragment peptidecomprises an amino acid sequence as set forth as SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.
 10. The method of claim 1,wherein the biological sample is a blood sample, a urine sample, a serumsample, an ascites sample, a sputum sample, lymphatic fluid, a salivasample, a cell, or a solid tissue.
 11. The method of claim 10, whereinthe tissue is formalin fixed tissue.
 12. The method of claim 10, whereinthe tissue is paraffin embedded tissue.
 13. The method of claim 10,wherein the tissue is obtained from a tumor. 14-16. (canceled)
 17. Themethod of claim 1, wherein quantifying the Her3 fragment peptidecomprises comparing an amount of one or more Her3 fragment peptidescomprising an amino acid sequence of about 8 to about 45 amino acidresidues of Her3 as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, or SEQ ID NO:10 in one biological sample to the amount of the sameHer3 fragment peptide in a different and separate biological sample. 18.The method of claim 17, wherein quantifying one or more Her3 fragmentpeptides comprises determining the amount of the each of the Her3fragment peptides in a biological sample by comparison to an addedinternal standard peptide of known amount, wherein each of the Her3fragment peptides in the biological sample is compared to an internalstandard peptide having the same amino acid sequence.
 19. The method ofclaim 18, wherein the internal standard peptide is an isotopicallylabeled peptide.
 20. The method of claim 19, wherein the isotopicallylabeled internal standard peptide comprises one or more heavy stableisotopes selected from 18O, 17O, 34S, 15N, 13C, 2H or combinationsthereof.
 21. The method of claim 1, wherein detecting and quantifyingthe amount of one or more modified or unmodified Her3 fragment peptidesin the protein digest indicates the presence of modified or unmodifiedHer3 protein and an association with cancer in the subject.
 22. Themethod of claim 21, further comprising correlating the results of saiddetecting and quantifying the amount of one or more modified orunmodified Her3 fragment peptides, or the level of said Her3 protein tothe diagnostic stage/grade/status of the cancer.
 23. The method of claim22, wherein correlating the results of said detecting and quantifyingthe amount of one or more modified or unmodified Her3 fragment peptides,or the level of said Her3 protein to the diagnostic stage/grade/statusof the cancer is combined with detecting and/or quantifying the amountof other proteins or peptides from other proteins in a multiplex formatto provide additional information about the diagnosticstage/grade/status of the cancer.
 24. The method of claim 1, furthercomprising selecting for the subject from which said biological samplewas obtained a treatment for the subject based on the presence, absence,or amount of one or more Her3 fragment peptides or the level of Her3protein.
 25. The method of claim 1, further comprising administering tothe patient from which said biological sample was obtained atherapeutically effective amount of a therapeutic agent, wherein thetherapeutic agent and/or amount of the therapeutic agent administered isbased upon the amount of one or more modified or unmodified Her3fragment peptides or the level of Her3 protein.
 26. The method of claim24, wherein said therapeutic agent binds the Her3 protein and/orinhibits its biological activity.
 27. The method of claim 25, whereinsaid therapeutic agent binds the Her3 protein and/or inhibits itsbiological activity.
 28. The method of claim 1, wherein the biologicalsample is formalin fixed tumor tissue that has been processed forquantifying the amount of one or more modified or unmodified Her3fragment peptides employing the Liquid Tissue™ protocol and reagents.29-33. (canceled)
 34. The method of claim 1, further comprisingassessing and/or determining the level (amount) or sequence of one, two,three, four or more nucleic acids in said protein digest.